In order to perform treatments using anionic drugs, particularly nucleic acid materials, safe and efficient drug delivery technologies have been studied for a long time, and various delivery systems and techniques have been developed. Particularly, delivery technologies employing viral delivery systems based on an adenovirus or a retrovirus, and non-viral delivery systems based on cationic lipids or cationic polymers have been developed.
However, it has been known that the technologies employing the viral delivery systems have problems in commercialization, including the risk of non-specific immune responses and the complexity in the production processes. For this reason, a recent research trend is to overcome the shortcomings of viral delivery systems by means of using non-viral delivery systems based on cationic lipids or cationic polymers. Such non-viral delivery systems are less efficient than viral delivery systems, but have the advantages of being accompanied by fewer side effects in vivo and having lower production costs.
Many studies have been conducted on non-viral delivery systems used for delivery of nucleic acid materials, and most typical examples thereof include a complex of cationic lipid and nucleic acid (lipoplex) and a complex of a polycationic polymer and nucleic acid (polyplex). This cationic lipid or polycationic polymer has been much studied, because it forms a complex by electrostatic interactions with an anionic drug, thereby stabilizing the anionic drug and increasing the intracellular delivery of the anionic drug (De Paula D, Bentley M V, Mahato R I, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56; Gary D J, Puri N, Won Y Y, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73).
However, if the cationic lipids or polycationic polymers developed to date are used in an amount required to obtain sufficient effects, they may cause serious toxicity—although less than that caused by viral delivery systems—indicating that they are unsuitable for therapeutic applications. In addition, a lipid-nucleic acid complex for intracellular delivery of the nucleic acid is widely used in cell line experiments, but it does not form a stable structure in blood, and thus it cannot be used in vivo (see U.S. Pat. No. 6,458,382).
Another non-viral delivery system that is used for the intracellular delivery of a nucleic acid in vivo is nucleic acid-cationic liposome complex or a cationic liposome containing a nucleic acid. It comprises an amphiphilic lipid, a neutral lipid, a fusogenic lipid, or the like, and the nucleic acid is electrically bound to the liposome or is entrapped in the liposome (US2003-0073640, WO05/007196, and US2006-0240093). However, this liposome delivery system may be easily captured by the reticuloendothelial system (RES) and show side effects with significant toxicity, indicating that it is unsuitable for systemic use. Also, the nucleic acid delivery effects thereof are mostly limited to liver tissue.
In addition, a non-viral delivery system that has been most frequently studied together with the liposome delivery system is a delivery system comprising a polycationic polymer containing a multivalent cationic charge per molecule. In this delivery system, a polymer that is frequently used is polycationic polyethyleneimine (PEI) based polymer, which is electrostatically bound to a nucleic acid to form a nanoparticle consisting of a nucleic acid-polymer complex. However, it is known that such polycationic polymers stimulate cell death and this cytotoxicity increases as the molecular weight and the degree of branching of the polymer increase. Also, it is known that polycationic polymers with low molecular weight have low cytotoxicity, but they cannot effectively form a complex with a nucleic acid due to their low cationic density, and thus they do not achieve the sufficient intracellular delivery of the nucleic acid and do not greatly contribute to the stability of the nucleic acid.
Another type of nucleic acid delivery system includes a delivery system obtained by conjugating a lipid or a polymer directly to a nucleic acid and then forming a complex of the conjugate with a micelle or another polymer to form a nanoparticle. However, conjugating the lipid or the polymer directly to the nucleic acid has difficulty in terms of conjugation efficiency or quality control, and the efficiency of nucleic acid delivery of this delivery system has not yet been clearly verified.
Therefore, it is required to develop an anionic drug delivery system in which the amount of cationic polymer or cationic lipid used can be minimized to reduce cytotoxicity and which is stable in blood and body fluid and can be delivered into cells to exhibit sufficient effects.
Meanwhile, there have been various attempts to use amphiphilic block copolymer to solubilize a poorly water-soluble drug in the form of a polymeric micelle and to stabilize the drug in an aqueous solution, thereby providing a drug delivery system (Korean Registered Patent No. 0180334). This amphiphilic block copolymer can solubilize a hydrophobic poorly water-soluble drug by forming polymeric micelles, but hydrophilic drugs such as anionic nucleic acids cannot be entrapped in the polymeric micelles, and thus the amphiphilic block copolymer is not suitable for delivery of these anionic drugs including nucleic acids. Thus, in order to deliver an anionic drug in the form of a polymeric micelle, a process of neutralizing the charge of the anionic drug using a cationic material is required. This process is disclosed in International Patent Publication No. WO 2010/074540.
Meanwhile, many diseases are caused by an increased expression of disease-related genes that occurs due to various factors or by abnormal activity caused by mutation. siRNA (short interfering RNA) inhibits the expression of a specific gene in a sequence-specific manner at the post-transcriptional stage, and receives a great deal of attention as a therapeutic agent. Particularly, due to its high activity and precise genetic selectivity, siRNA is expected as a therapeutic agent that can substitute for existing antisense nucleotides or ribozymes. siRNA is a short double-stranded RNA molecule composed of 15-30 nucleotides and cleaves the mRNA of a gene having a nucleotide sequence complementary thereto to inhibit the expression of the gene (McManus and Sharp, Nature Rev. Genet. 3:737 (2002); Elbashir, et al., Genes Dev. 15:188 (2001)).
Despite such advantages, siRNA is known to be rapidly degraded by nucleases in the blood and to be rapidly excreted through the kidney. In addition, it is known that siRNA does not easily pass through the cell membrane because it is negatively charged. For this reason, in order to use siRNA as a therapeutic agent, it is required to develop a technology for preparing a system for delivering siRNA, which can stabilize siRNA in vivo, deliver siRNA into a target cell or a target organ efficiently and show no toxicity.